Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF.

Journal article

The porins OmpF and OmpC are trimeric β-barrel proteins with narrow channels running through each monomer that exclude molecules > 600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer membrane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (> 6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B(12) receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured translocation domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpF-binding sites, OBS1 (residues 2-18) and OBS2 (residues 54-63), which flank the TBE and bind with K(d)s of 2 and 24 μM, respectively, at pH 6.5 and 25 ºC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow cross-sectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a β-barrel membrane protein.

Authors: Housden, N; Wojdyla, J; Korczynska, J; Grishkovskaya, I; Kirkpatrick, N

• Publication status: Published
• Journal: Proceedings of the National Academy of Sciences of the United States of America
• Volume: 107
• Issue: 50
• Pages: 21412-21417
• Publication date: 2010-12-01
• DOI: 10.1073/pnas.1010780107
• EISSN: 1091-6490
• ISSN: 0027-8424
• Language: English
• Keywords: Colicins; Crystallography, X-Ray; Models, Molecular; Recombinant Fusion Proteins; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Protein Structure, Tertiary; Epitopes; Porins; Cell Membrane